Centrifugation has often been employed as a separation technique. In many fields, such as genetic engineering, materials are separated by centrifugation and sedimentation within a cesium chloride or other density-type gradient. After centrifugation and sedimentation, fractions of the centrifuge tube are removed and analyzed. The density of a substance determines where within the cesium chloride gradient the substance settles. This position within the gradient can be specified in terms of a distance from the center of rotation. The density of the substance can be determined by knowing the gradient and the distance from the center of rotation at which the substance settled. Thus, not only can substances of varying densities be separated by this method, but accurate density determinations may also be made.
From the above discussion, it can be understood that the degree of separation achieved, or the precision within which the density of a substance can be determined, is dependent upon the degree to which fractions (or layers) can be removed from the centrifuge tube for analysis without mixing between the layers.
One apparatus disclosed for sequential fractionation is described by Chervenka et al in U.S. Pat. No. 4,181,700. The device include a microsyringe mounted to a movable frame and a suction means for withdrawing fluid from the centrifuge tube into the syringe. The syringe is lowered a precise distance into a centrifuge tube and this distance is read from a micrometer and recorded. Suction is then applied to the syringe tip to remove a precise volume of liquid from the top of the centrifuge tube. While the above method is tolerable for many applications, serious difficulties arise if high precision is desired.
As stated above, precision is related to the degree of mixing which occurs between layers. When suction is applied through the syringe, flow occurs within the centrifuge tube. Since laminar flow laws apply, it is clear that liquid at the center of the tube flows faster than liquid at the outer edges. Thus, a significant amount of mixing inherently occurs.
Another apparatus (U.S. Pat. No. 3,151,639 to Allington) sequentially removes layers from a centrifuge tube by forcing a dense liquid into the bottom of the centrifuge tube to raise the level of the other liquid in the tube an amount corresponding to the volume of the added dense liquid. The liquid in the centrifuge to is forced out of the tube and into a fraction collector solely by the action of the added dense liquid. Although the application of suction is avoided by this method, large amounts of laminar flow and thus mixing still occur, since each time dense fluid is added, the entire liquid mass within the centrifuge tube must move upwardly.